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Appl Environ Microbiol, January 1998, p. 14-20, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Production of Active Chimeric Pediocin AcH in Escherichia coli in the Absence of Processing and Secretion Genes from the Pediococcus pap Operondagger

Kurt W. Miller,1,* Robin Schamber,1 Yanling Chen,2 and Bibek Ray2,*

Departments of Animal Science2 and Molecular Biology,1 University of Wyoming, Laramie, Wyoming 82071

Received 26 June 1997/Accepted 12 October 1997

Minimum requirements have been determined for synthesis and secretion of the Pediococcus antimicrobial peptide, pediocin AcH, in Escherichia coli. The functional mature domain of pediocin AcH (Lys+1 to Cys+44) is targeted into the E. coli sec machinery and secreted to the periplasm in active form when fused in frame to the COOH terminus of the secretory protein maltose-binding protein (MBP). The PapC-PapD specialized secretion machinery is not required for secretion of the MBP-pediocin AcH chimeric protein, indicating that in Pediococcus, PapC and PapD probably are required for recognition and processing of the leader peptide rather than for translocation of the mature pediocin AcH domain across the cytoplasmic membrane. The chimeric protein displays bactericidal activity, suggesting that the NH2 terminus of pediocin AcH does not span the phospholipid bilayer in the membrane-interactive form of the molecule. However, the conserved Lys+1-Tyr-Tyr-Gly-Asn-Gly-Val+7-sequence at the NH2 terminus is important because deletion of this sequence abolishes activity. The secreted chimeric protein is released into the culture medium when expressed in a periplasmic leaky E. coli host. The MBP fusion-periplasmic leaky expression system should be generally advantageous for production and screening of the activity of bioactive peptides.

* Corresponding author. Mailing address for Kurt W. Miller: Department of Molecular Biology, P.O. Box 3944, University of Wyoming, Laramie, WY 82071-3944. Phone: (307) 766-2037. Fax: (307) 766-5098. E-mail: kwmiller{at}uwyo.edu. Mailing address for Bibek Ray: Department of Animal Science, P.O. Box 3684, University of Wyoming, Laramie, WY 82071-3684. Phone: (307) 766-3140. Fax: (307) 766-2350. E-mail: labcin{at}uwyo.edu.

dagger This paper is dedicated to the memory of Henry C. Wu.




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